Arrowhead (Awh) function is related to special developmental structures termed histoblast nests, precursors of certain abdominal structures in the adult fly. Unlike the process in vertebrate development, the adult fly is not formed as a result of the continuous development of embryonic tissues, rather imaginal cells from which various adult structures eventually arise are set apart from embryonic tissues early in embryonic development. Imaginal precursor cells are established as discrete groups of cells localized to specific regions of the embryo. The precursors of the adult head structures, appendages and genitalia form from invaginations of the embryonic epithelium and make up the imaginal discs; these are groups of cells not directly associated with the larval integument. The precursors of the abdomen and the internal organs of the adult, such as the gut, salivary glands and brain, arise from nests or rings of cells intimately associated with larval structures. For example, the salivary gland imaginal rings are embedded in the larval salivary glands; the midgut imaginal histoblast nests arise in the larval midgut and the abdominal histoblast nests form among the cells of the larval abdomen (Curtiss, 1995).

Each adult abdominal segment forms from four pairs of histoblast nests: the anterior and posterior dorsal pairs (which produce the tergites); the ventral pair (which produce the sternites and pleurites), and the spiracular pair (which form the spiracle and the surrounding pleurite tissues). Each anterior dorsal and ventral histoblast nest is composed of approximately 16 cells; each posterior dorsal histoblast nest consists of approximately five cells, and each spiracle histoblast nest has approximately three cells. The abdominal histoblasts do not divide during the larval stages, but begin to divide within the first 3 hours after pupariation. They continue to divide until approximately 15 hours of pupal development without displacing the larval cells. At about 15 hours of pupal life, the abdominal histoblast cells begin to migrate and displace the larval cells, which are then histolyzed. Following proliferation and migration, cells of adjacent segments fuse at the dorsal/ventral and segmental borders. During the terminal stages of abdominal development the cells differentiate to produce epidermal tissues, including the microchaetae and macrochaetae, and tosecrete the adult cuticle (Curtiss, 1995 and references).

In the Awh mutant pharate adult, a single row of bristles develop in the anterior-most segment. No other development of the abdominal epithelium occurs, as evidence by the absence of bristles and cuticle. Nevertheless, when partial development of abdominal epithelium occurs in mutant pupae, the cuticle and bristles appear normal. It has been concluded that Awh does not affect differentiation of the cells, but does affect the establishment or proliferation of the precursors. Examination of escargot (a gene required for cell cycle regulation of imaginal tissue) expression in abdominal histoblasts and other imaginal precursors shows that Awh mutants have significantly fewer cells in each histoblast nest. This suggests that Awh is necessary to generate the proper number of abdominal histoblasts in the embryo (Curtiss, 1995).

Awh appears to have a role in salivary gland formation as well. The adult salivary glands develop from imaginal rings located at the anterior end of each larval salivary gland. The imaginal ring cells resume mitosis at the molt between second and third instar. The larval salivary gland degenerates during the early pupal stages until, by approximately 24 hours of pupal development, it is completely dissolved. At approximately 10 hours of pupal development the imaginal ring cells begin to grow out anteriorly and posteriorly to form the adult salivary gland. Approximately 10 cells in each ring of wild-type late second instar constitute the embryonic founder cells of the salivary gland imaginal ring. Each imaginal ring contains approximately 150 cells in mature third instar larvae. In Awh mutant larvae, imaginal ring cells are often completely absent or reduced in number, and their arrangement is disorganized. Thus Awh is required for development of both abdominal histoblasts and salivary gland imaginal ring cells (Curtiss, 1995).

Expression of Awh in histoblast and imaginal ring tissue, and the requirement for Awh for the proliferation of these tissues, points to a clear distinction between two types of imaginal tissues: (1) imaginal discs that give rise to adult structures such as wings, legs and gonads do not require Awh; this is in contrast to (2) histoblasts and imaginal ring tissue that do require Awh function for establishment or proliferation. Curtiss and Helwig (1995) define as incorporate those imaginal precursor cells, including the abdominal histoblasts and salivary gland imaginal rings, that are embedded in larval tissue. During metamorphosis, incorporate imaginal cells replace the cognate larval organ in which the precursor cells are located. excorporate imaginal precursor cells are defined as imaginal discs, which develop separately from larval larval tissue. During metamorphosis, excorporate imaginal cells elaborate structures unique to the adult.

Several experimental results point to a clear distinction between these two types of imaginal tissue. Whereas expression of Awh is required for the development of incorporate imaginal precursors, ectopic expression of Awh promotes programmed cell death in excorporate imaginal cells. Expression of Awh in the wing disc in regions that give rise to the adult wing blade and part of the wing hinge give rise to adult flies that are missing all structures which derive from the portions of the imaginal discs that ectopically express Awh. However, ectopic expression of Awh in developing salivary glands results in normal functional tissues. A mutant allele, Awh1, a spontaneous gain-of-function allele that initially defined the Awh locus, exhibits no defect other than a reduction in size for compound eyes. In Awh1 mutant eyes, genes properly expressed in the developing eye (such as hairy, decapentaplegic, scabrous and glass), fail to be expressed in patterns corresponding to compoud eye development. Since the compound eye develops normally in Awh mutant pharate adults, it is not believed that Awhplays a requisite role in retinal development. The phenotype of the neomorphic Awh1 mutation is likely the result of altered expression of Awh that interferes with retinal development. This phenotype resembles that derived from ectopic expression of Awh in imaginal discs (Curtiss, 1995 and Curtiss, 1997).

Other evidence points to a significant difference between excorporate and incorporate imaginal tissue. The two types of tissues exhibit different modes of cell proliferation control. string, a mitotic inducer that encodes a fly homolog of cdc25 phosphatases, is essential for the G2 phase to mitosis transition in the 14th cell cycle during embryogenesis. string is also essential for the generation of imaginal tissue. Clones of a strong mutant allele were generated in growing imaginal tissue. Normal bristle formation is prevented by induction of string clones, indicating that string is essential for the generation of the adult cuticle, the tissue generated by division of histoblast nests. String's role in cell cycle regulation is illustrated by the observation that ectopic expression of string in the imaginal discs of the thorax induces mitoses in G2-arrested cells. Ectopic String also induces premature and extra mitoses in the developing eye-antennal imaginal disc. In contrast to the ability of String to induce mitosis in imaginal discs, imaginal histoblasts prove to be refractory to ectopic String. These experiments suggest that in wandering third istar larvae, a factor or factors other than String are limiting for entry of abdominal histoblasts into mitosis This is the first example of G2 arrest during fly development that is not mediated by string transcriptional regulation (Kylsten, 1997).

It is concluded that Arrowhead expression is required for development of one set of imaginal cells,
the incorporate cells that are embedded in larval tissue, and is incompatible with development of another, the excorporate cells that develop as imaginal discs. Future work could elucidate a mechanism whereby Arrowhead functions as an upstream regulator of cell cycle.

WNK signaling is involved in neural development via Lhx8/Awh expression

WNK kinase family is conserved among many species and regulates SPAK/OSR1 and ion co-transporters. Some mutations in human WNK1 or WNK4 are associated with Pseudohypoaldosteronism type II, a form of hypertension. WNK is also involved in developmental and cellular processes, but the molecular mechanisms underlying its regulation in these processes remain unknown. This study identified a new target gene in WNK signaling, Arrowhead and Lhx8, which is a mammalian homologue of Drosophila Arrowhead. In Drosophila, WNK was shown to genetically interact with Arrowhead. In Wnk1 knockout mice, levels of Lhx8 expression were reduced. Ectopic expression of WNK1, WNK4 or Osr1 in mammalian cells induced the expression of the Lhx8. Moreover, neural specification was inhibited by the knockdown of both Wnk1 and Wnk4 or Lhx8. Drosophila WNK mutant caused defects in axon guidance during embryogenesis. These results suggest that WNK signaling is involved in the morphological and neural development via Lhx8/Arrowhead (Sato, 2013).

The WNK-SPAK/OSR1 pathway is known to regulate various ion co-transporters and is widely conserved among many species. Wnk1 knockout mice die before embryonic day 13, and display defects in cardiac development. WNK1 is also required for cell division in cultured cells. Furthermore, PHAII patients display a number of other clinical features, such as an intellectual impairment, dental abnormalities and impaired growth in addition to hypertension. Accordingly, the new role of the WNK signaling pathway described in this study may provide further insight into the development and pathogenesis of PHAII. In this study, Lhx8/Awh was identifed as a new downstream molecule in the WNK-SPAK/OSR1 pathway, and a novel function was discovered for the WNK-Lhx8 pathway in neural development (Sato, 2013).

There are four mammalian WNK family members, and WNK1 and WNK4 genes are linked to a hereditary form of human hypertension known as Pseudohypoaldosteronism type II (PHAII). In Drosophila, only one WNK gene, DWNK, has been identified. This study found that both the wild-type and kinase-dead forms of WNK1 or WNK4 caused the up-regulation of Lhx8 gene expression in NIH3T3 cells. Similarly, a previous study showed that SPAK, a substrate of WNK1, was weakly phosphorylated by the kinase-dead form of WNK1 following a long incubation (Moriguchi, 2005). These results are inconsistent with the idea that the kinase-dead form of DWNK functions as a dominant-negative mutant in Drosophila. Studies of WNK1 and WNK4 suggest that these molecules phosphorylate each other and coordinated to regulate NaCl cotransport. Therefore, these results raised the possibility that the kinase-dead forms of WNK1 and WNK4 coordinate with their respective endogenous WNK1 and WNK4 counterparts in mammalian cells. In fact, this study found that co-expression of both kinase-dead forms of WNK1 and WNK4 did not cause either induction of Lhx8 gene expression or phosphorylation of mOsr1. These results suggest that the kinase activity of WNKs is required for induction of Lhx8 gene expression and the activation of SPAK/OSR1, and that the kinase-dead form of WNK acts as an actual dominant-negative form in the signaling pathway. Furthermore, the expression of Lhx8 by either hypertonic or RA stimulation was required for the expression of both WNK1 and WNK4. Taken together, these results suggest that WNK1 and WNK4 function coordinately and redundantly in mammalian cells (Sato, 2013).

A previous report demonstrated that WNK1 might control the formation of microtubules in developing neurons. Other studies suggested that Lhx8 plays an important role in the development of basal forebrain cholinergic neurons, that Fray is required for axonal ensheathment, and that Awh is expressed in neuroblasts in stage 9 embryos in Drosophila. This study showed that the WNK-OSR1 pathway regulates Lhx8 gene expression, that knockdown of both Wnk1 and Wnk4 in Neuro2A cells caused a shortening of neurites, as well as reduced Lhx8 expression, and that the expression of the constitutively active form of mOsr1, mOsr1S325D, could rescue the phenotype caused by the knockdown of both Wnk1 and Wnk4. In addition, mutation of DWNK or expression of a dominant-negative form of DWNK in fly embryos caused defects in axon guidance in the peripheral nervous system, and the constitutively active form of fray, frayS347D, expression could rescue the phenotypes by the expression of the dominant negative form of DWNK. Furthermore, ubiquitous expression of Awh by da-Gal4 showed severe defects of axon guidance as similar to DWNKD420A expression by da-Gal4, although neural specific expression of Awh did not showed any phenotype. Taken together, these findings clearly indicate that the WNK-OSR1/Fray-Lhx8/Awh pathway is involved in neural development. However, the phenotypes caused by knockdown of both Wnk1 and Wnk4, such as the shortening of neurites and the reduction in ChAT expression, were not rescued by the expression of Lhx8 in Neuro2A cells. In addition, the expression of Awh could not rescue the defects in the peripheral nervous system by the expression of the dominant-negative form of DWNK. Previous reports showed that Lhx8 might work with other factors, such as Lhx6 or Isl1. However, this study also found that the expression of Lhx6 and/or Isl1 with Lhx8 could not rescue the defects by knockdown of both Wnk1 and Wnk4 in Neuro2A cell. These results suggest that other molecule(s) are involved in neural differentiation induced by WNK signaling. The current studies may provide the first evidence identifying a target gene that acts downstream in the WNK-SPAK/OSR1 pathway, and demonstrate the significance of the WNK-OSR1-Lhx8 pathway in neural development. However, the details of how other unknown molecules controlled by WNK signaling specifically contribute to neural developmental remain to be determined and will require additional study (Sato, 2013).

Genetic mutations of WNK1 or WNK4 in PHAII patients result in abnormal expression of the WNK1 gene or WNK4 kinase activity, respectively. Abnormal activation of the WNK signaling pathway caused by these mutations result in the misregulation of NCCs in the kidney, which in turn causes hypertension. However, PHAII patients display other clinical features, such as an intellectual impairment, dental abnormalities and impaired growth. Although these features are also thought to be caused by WNK1 or WNK4 mutations, the details of how these pathologies occur are unknown except for hypertension. This study identified Lhx8 as a downstream target of the WNK signaling pathway. Evidence was also found that the WNK-Lhx8 pathway is involved in neural development. Previous studies have shown that knockdown of Lhx8 using antisense oligodeoxynucleotides caused the loss of tooth germ, and Lhx8 and Lhx6 are key regulators of mammalian dentitio. Furthermore, Lhx8 knockout mice show a reduction in the number of cholinergic neurons in the ventral forebrain and exhibit a severe deficit in spatial learning and memory . These observations indicate that Lhx8 has essential functions in the formation of the tooth development, the specification of the cholinergic neurons and the processing of the spatial information in mice. Therefore, the similarities between the clinical features of PHAII and the phenotypes of Lhx8 knockdown or knockout mice strongly suggest that the WNK-Lhx8 pathway is involved in the pathogenesis of PHAII, aside from hypertension. Further investigation will be needed to prove this hypothesis (Sato, 2013).

Awh transcription is first detected in cells located at the position of the neuroblasts in stage 9 embryos, when neuroblasts begin to segregate. Loss-of-function mutations in the neurogenic gene Delta result in hypertrophy of the nervous system. Embryos carrying a loss-of-function mutation in Delta have about twice as many Awh-expressing cells as wild-type embryos, suggesting that the Awh expressing cells are neuroblasts. Awh is also transcribed at stage 11 in cells in the procephalon that may correspond to cells of the supraesophageal ganglia (brain). During embryogenesis, Arrowhead is expressed in each abdominal segment
and in the labial segment, consistent
with its role in establishing the proper numbers of abdominal histoblasts and salivary gland imaginal ring cells (Curtiss, 1995). AWH mRNA expression during stage 10 of embryogenesis, the extended germ band stage, is found in bilateral stripes in the three thoracic segments and in all 10 abdominal segments. As the gnathal buds become apparent during stage 11, the Awh transcript is also detected in a stripe and a few additional cells at the center of the labial bud. At stage 14, Awh is not expressed in cells in the region of the thoracic segments in which the developing prothoracic, wing, haltere, and leg discs are located. escargot, a gene involved in imaginal disc cell cycle regulation. is expressed in the developing discs from which Awh expression is excluded. Late in embryonic development, expression is refined to the abdominal
histoblasts and salivary gland imaginal ring cells themselves (Curtiss, 1997).

Metamorphosis in Drosophila melanogaster requires synchronization of numerous developmental
events that occur in isolated imaginal precursor tissues. The imaginal primordia are established during
embryonic stages and are quiescent for much of larval life. The Arrowhead gene is necessary for the
establishment of proper numbers of cells within a subset of imaginal precursor tissues. Loss-of-function
mutations in Arrowhead reduce the number of abdominal histoblasts and salivary gland imaginal ring
cells before the proliferative stages of their development. The number of abdominal histoblasts in
mutant animals is approximately half that of wild-type, as might result from failure of a single early
division of these cells. A neomorphic Arrowhead allele, Awh1, results in the specific loss of the retinal
precursors by the early third instar, before they have begun to differentiate. Since Arrowhead
mutations affect only subsets of imaginal tissue, there must be distinctions in the developmental
regulation of different imaginal precursors. Arrowhead may be part of a regulatory pathway
responsible for establishing the proper number of abdominal histoblasts and salivary gland imaginal ring
cells. The neomorphic Arrowhead allele, which may cause misexpression of the Arrowhead gene in
the eye-antenna imaginal disc, interferes with the establishment or proliferation of retinal precursor
cells (Curtiss, 1995).

The transcriptional co-factor Chip acts with LIM-homeodomain proteins to set the boundary of the eye field in Drosophila

Development involves the establishment of boundaries between fields specified to differentiate into distinct tissues. The Drosophila larval eye-antennal imaginal disc must be subdivided into regions that differentiate into the adult eye, antenna and head cuticle. The transcriptional co-factor Chip is required for cells at the ventral eye-antennal disc border to take on a head cuticle fate; clones of Chip mutant cells in this region instead form outgrowths that differentiate into ectopic eye tissue. Chip acts independently of the transcription factor Homothorax, which was previously shown to promote head cuticle development in the same region. Chip and its vertebrate CLIM homologues have been shown to form complexes with LIM-homeodomain transcription factors, and the domain of Chip that mediates these interactions is required for its ability to suppress the eye fate. Two LIM-homeodomain proteins, Arrowhead and Lim1, are shown to be expressed in the region of the eye-antennal disc affected in Chip mutants, and both require Chip for their ability to suppress photoreceptor differentiation when misexpressed in the eye field. Loss-of-function studies support the model that Arrowhead and Lim1 act redundantly, using Chip as a co-factor, to prevent retinal differentiation in regions of the eye disc destined to become ventral head tissue (Roignant, 2009).

Regionalization of the eye-antennal disc is a progressive process in which selector genes and signaling pathways specify the fates of different head structures. Clones of eye-antennal disc cells induced during the second larval instar can contribute to multiple organs, indicating that these cells retain developmental plasticity at this stage. The anteroposterior boundary of the wing disc is established much earlier; expression of the selector gene engrailed (en) specifically in the posterior cells during embryogenesis generates an affinity border that keeps the two compartments clonally separated. By contrast, the eye selector gene ey is uniformly expressed throughout the early eye-antennal disc, and only retracts to the eye field in the second instar. It was initially proposed that localized Notch signaling controls this retraction, as expression of dominant-negative forms of Notch in the eye disc abolishes ey expression and leads to antennal duplications. However, a later study demonstrated that loss of Notch function does not affect ey expression directly, but reduces cell proliferation in the retinal field, preventing the initiation of eya expression. This study shows that Chip and Lim1 are both necessary to repress ey expression in the anterior of the antennal disc. Additional factors probably help to restrict ey expression to the eye disc, because ey expression does not extend throughout the normal Lim1 expression domain in Lim1 or Chip mutant clones in the antennal disc (Roignant, 2009).

Since Lim1 mutant clones always misexpress Ey, but rarely misexpress Eya and never differentiate ectopic photoreceptors, additional proteins must interact with Chip to repress retinal differentiation. Awh is a good candidate because it is expressed at the ventral margin of the eye-antennal disc, its misexpression in the retina represses photoreceptor differentiation in a Chip-dependent manner, and loss of both Lim1 and Awh leads to ectopic photoreceptor differentiation in the ventral eye-antennal disc. Since ectopic photoreceptors differentiate only in the absence of both Lim1 and Awh, whereas Ey expansion is observed in Lim1 single mutants, Awh must control the expression of target genes other than ey. It may negatively regulate other genes involved in retinal determination, such as eya, or positively regulate genes important for head capsule development, such as Deformed and odd-paired (Roignant, 2009).

Like Chip, Hth is required to prevent retinal differentiation at the ventral eye-antennal disc boundary. Investigation of the relationship between Chip and Hth indicates that Chip is not required for Hth expression or activity. The ability of Hth to repress photoreceptor differentiation in Chip mutant clones rules out the possibility that Chip acts as a co-factor for Hth or an essential downstream mediator of its effects. The normal expression of Hth and its target gene wg in Chip mutant clones also make it unlikely that Chip controls the expression of Hth or its co-factor Exd. However, the possibility that Hth and Chip act in parallel poses the paradox that misexpressed Hth is sufficient to repress photoreceptor development in the eye field in the absence of Chip, but endogenous Hth is insufficient to do so in the head field. It is possible that Hth expression levels in the head field early in development are too low to repress the eye fate in the absence of Chip. Consistent with this hypothesis, it was found that overexpression of Hth in Chip mutant cells prevents ectopic photoreceptor differentiation. Similarly, overexpression of Awh or Lim1 prevents ectopic photoreceptor differentiation in hth mutant cells, suggesting that endogenous levels of these LIM-HD proteins are not sufficient to compensate for the absence of Hth. The two classes of transcription factors may normally act on different sets of target genes, but show some cross-regulatory ability when overexpressed (Roignant, 2009).

The boundary between the eye and the dorsal head appears to be established differently from the boundary in the ventral region. The LIM-HD gene tup is expressed at the dorsal eye-antennal disc boundary, in a pattern resembling the mirror image of the Awh pattern, and is capable of repressing photoreceptor development in a Chip-dependent manner. However, loss of Chip in this region does not lead to ectopic eye formation, although it can cause overgrowth and mispatterning of the head. In the absence of Chip, the GATA transcription factor Pannier (Pnr) and its target gene wg may be sufficient to maintain dorsal head fate. The ventral margin of the eye-antennal disc may be particularly susceptible to ectopic photoreceptor differentiation because of the high level of Dpp signaling there. A 5' enhancer element has been shown to direct dpp expression specifically in the ventral marginal peripodial epithelium of the eye-antennal disc. The ability of Dpp and Ey to synergize to drive retinal differentiation therefore makes it critical to repress Ey in this region, which is fated to form head capsule (Roignant, 2009).

In addition, this domain of Dpp overlaps with Wg present at the anterior lateral margin of the eye disc; the combination of these two growth factors induces proximodistal growth of the leg. One function of Chip and its partner proteins might thus be to repress the outgrowth that would otherwise be triggered by the combination of Dpp and Wg. Unlike growth of the wild-type eye disc, growth of Chip mutant regions appears to be Notch-independent, as they do not contain a fng expression boundary and do not show activation of the Notch target genes E(spl)mβ or eyg. Notch has been thought to trigger growth by inducing the expression of the JAK/STAT ligand Unpaired (Upd); however, a recent report describes an earlier function for Upd upstream of Notch, raising the possibility that upd expression is activated independently of Notch in Chip mutant clones. As hth mutant clones, or clones lacking the Odd skipped family member Bowl, frequently show ectopic ventral photoreceptor differentiation but rarely induce outgrowths like those seen in Chip mutants, the functions of Chip in growth and differentiation are likely to be separable (Roignant, 2009).

LIM-HD proteins also set developmental boundaries in other imaginal discs, acting in concert with other classes of transcription factors. In the wing disc, Tup specifies the notum in collaboration with homeodomain transcription factors of the Iroquois complex, and Ap specifies the dorsal compartment. Ap interacts with the homeodomain protein Bar and Lim1 with Aristaless to establish specific tarsal segments within the leg disc. LIM-HD proteins have also been implicated in vertebrate eye development, although those that have been studied appear to play positive roles. The Ap homologue Lhx2 is expressed within the mouse retinal field at the neural plate stage, and contributes to the expression of Pax6, Six3 and Rx. Lmx1b, the homologue of CG32105, is required for the development of anterior eye structures such as the cornea and iris, and is mutated in human patients with nail-patella syndrome, often characterized by glaucoma. Within the retina, loss of Lim1 results in mispositioning of horizontal cells within the amacrine cell laye. Drosophila Lim3 shows photoreceptor-specific expression, and might therefore have a positive function in eye development (Roignant, 2009).

In the central nervous system, LIM-HD proteins act combinatorially to specify different neuronal cell fates. In both Drosophila and vertebrates, combinations of Islet and Lhx3/4/Lim3 proteins regulate motoneuron specification and pathfinding. The ability of Chip to interact with LIM-HD proteins and other transcription factors as well as to dimerize enables it to form heteromeric transcription factor complexes. In the wing disc, the active complex is a tetramer containing two subunits each of Chip and Ap, whereas in motoneuron development the Chip homologue NLI can form either a tetramer with Lhx3 or a hexamer containing both Isl1 and Lhx3. The finding that Lim1 and Awh act redundantly to prevent eye development in the ventral head primordium, whereas Chip is absolutely required, seems most consistent with regulation of distinct subsets of target genes by independent Chip-Awh and Chip-Lim1 complexes; however, a contribution from a complex containing all three proteins, or even additional transcription factors, cannot be ruled out. The role of the Chip co-factor may be to coordinate multiple transcriptional regulatory complexes to restrict developmental fates within the eye-antennal imaginal disc, allowing it to give rise to the head cuticle as well as distinct external sensory structures (Roignant, 2009).

EVOLUTIONARY HOMOLOGS

Awh has two LIM domains and a homeodomain. The LIM-2 domain of Awh differs from that of other members of the family in that the first and second cysteines are separated by three residues, rather than the usual two. The atypical spacing of these cysteines in Awh LIM-2 is not likely to affect the overall structure of the domain. The protein can be thought of as being divided into three parts. The LIM-1 domain stretches from amino acids 8 through 60; the LIM-2 domain from amino acids 69 through 122, and the homeodomain from amino acids 152 through 207. Awh has no identifiable close affinities to Islet or Apterous type LIM homeodomain proteins; outside the LIM- and homeo-domains, the protein contains no significant similarity to other proteins currently in the database (Curtiss, 1997).

The C. elegans AWA, AWB, and AWC olfactory neurons are each required for the recognition of a specific subset of volatile odorants. lim-4 mutants express an AWC reporter gene inappropriately in the AWB olfactory neurons and fail to appropriately express an AWB reporter gene. The AWB cells are morphologically transformed toward an AWC fate in lim-4 mutants, adopting cilia and axon morphologies characteristic of AWC. AWB function is also transformed in these mutants: Rather than mediating the repulsive behavioral responses appropriate for AWB, the AWB neurons mediate attractive responses, like AWC. LIM-4 is a predicted LIM homeobox gene that is expressed in AWB and a few other head neurons. Ectopic expression of LIM-4 in the AWC neuron pair is sufficient to force those cells to adopt an AWB fate. The AWA nuclear hormone receptor ODR-7 also represses AWC genes, as well as inducing AWA genes. It is proposed that the LIM-4 and ODR-7 transcription factors function to diversify C. elegans olfactory neuron identities, driving them from an AWC-like state into alternative fates (Sagasti, 1999).